Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020–2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.


SEQUENCING AND GENOME ASSEMBLY
Complete influenza A virus genomes were sequenced with the Illumina MiSeq platform by FFA and Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) and MinION Sequencing device (Oxford Nanopore) platform by Finnish Institute for Health and Welfare (THL).PCR amplicons were produced using the Uni12/Inf1, Uni12/Inf3, Uni13/Inf1 primers [1,2].For Illumina Miseq platform, sequencing libraries were obtained using the Nextera XT DNA Library Preparation Kit (Illumina) and quantified using Qubit (Invitrogen, USA).The average fragment length was determined using the Agilent High Sensitivity Bioanalyzer Kit.The indexed libraries were pooled in equimolar concentrations and sequenced using the MiSeq Reagent Micro Kit v2 (300-cycles, Illumina).For Oxford Nanopore sequencing, Rapid Barcoding Kit 96 (SQK-RBK110-96) was used with either 5 ul or 7,5 ul of sample.About 700 ng of library was loaded into a MinION Sequencing device and run for up to 24 hours.In addition, the sequencing at the IZSVe was performed on an Illumina MiSeq platform as previously described [3].
The whole genome assembly was performed at FFA, IZSVe and the Worldwide Influenza Centre (WIC), Francis Crick Institute (London, UK).At FFA, the sequence data was analysed using CLC Genomics Workbench (Qiagen).Raw reads were quality trimmed and adapter sequences were removed and trimmed reads were mapped against a reference sequence.The consensus sequences were generated with a minimum coverage depth of 6.At IZSVe, data was analysed using an in-house pipeline [1].After cleaning and trimming, high quality reads were aligned against a reference genome using BWA v0.7.1229.Alignments were processed with Picard tools v2.1.0(http://picard.sourceforge.net)and GATK v3.530-32 to correct potential errors, realign reads around indels and recalibrate base quality.Single nucleotide polymorphisms (SNPs) were called using LoFreq v2.1.233,and the outputs were used to generate consensus sequences.At the WIC, genome assembly was performed with the CDC IRMA pipeline, with a minimum depth of 6 [4].

GROSS PATHOLOGY
Most of the infected animals had multifocal to coalescing, variably sized haemorrhagic areas extending from the pleural surface to deep lung parenchyma.Some had petechial haemorrhages on the surface of the lungs, bloody fluid or extensive mucus in the trachea and mild to moderate amounts of clear to bloody pleural or peritoneal effusion.Spleens were mildly to markedly enlarged and dark.In most animals the liver was either diffusely pale or mottled or there were some petechial haemorrhages on the capsule.Some animals had mildly to markedly congested meninges.Many animals had empty stomachs and some mucoid or loose contents in the intestines.

LUNG
Majority (27/33) of both foxes and minks exhibited a multifocal to diffuse necrosuppurative bronchointerstitial pneumonia.6 animals had also diffuse acute fibrinoid pleuritis.26/33 of the animals had multifocal areas with large amounts of activated alveolar macrophages, plump highly activated type II pneumocytes and alveolar hyaline membranes consistent with diffuse alveolar damage.7 animals had moderate amounts of secondary bacterial colonies in the alveoli.A few (8/33) animals additionally had multifocal small randomly scattered interstitial mineralizations and 12 animals had mild multifocal erosions of tracheal epithelium.

BRAIN
Out of 19 animals that had brain histologically sampled, 14 had brain lesions multifocally in the cerebellar grey and white matter.They exhibited a mild to moderate multifocal subacute necrotizing meningoencephalitis with multifocal mononuclear perivascular cuffing and scattered small hemorrhages.

LIVER
The livers of 24/33 infected animals had mild to severe multifocal to coalescing periportal to midzonal or occasional random necrosis with occasional variable sized multifocal mineralizations.

SPLEEN
In spleens, 11 foxes and 3 minks had mild to severe depletion of lymphoid tissue with replacement of myriad of macrophages and multifocal hemorrhages.Most foxes and minks had moderate to severe congestion in the spleen and the splenic tissue of minks was spared with moderate to marked increase in megakaryocytes.
Out of 7 animals that had lymph nodes histologically sampled, 5 had lymph nodes mildly to moderately considered as reactive with activated secondary lymphoid follicles, mildly increased amounts of plasma cells and a medullary influx of macrophages.

INTESTINE
19 foxes had a diffuse, acute fibrinoid peritonitis.14/33 of all animals had mild multifocal cryptal epithelial cell necrosis in small or large intestine.

BACTERIOLOGY AND ADDITIONAL VIROLOGY
Most of the autopsied animals had variable bacterial growth in different organs.Detected bacteria (Streptococcus sp., Staphylococcus sp., Escherichia coli, Pasteurella sp. and Campylobacter sp.) are commonly found in fur animal samples in Finland.
Samples for canine distemper virus were taken as pooled samples per farm from the necropsied animals.Samples for parvovirus were taken from those animals that had diarrhea.Samples for canine adenovirus were taken from two foxes and SARS-CoV-2 samples were taken from the necropsied minks.All the virus assays for canine distemper virus, parvovirus, canine adenovirus and SARS-CoV-2 were negative.

EVOLUTIONARY ANALYSIS
Supplemental Table 1.The Bayesian factors and posterior probabilities of virus transmission events between the wild birds and farms that were identified in the evolutionary analysis.